Yeast cluster

Braymer, J. J.; Knauer, L.; Crack, J. C.; Oltmanns, J.; Heghmanns, M.; Soares, J. C.; Le Brun, N. E.; Schünemann, V.; Kasanmascheff, M.

doi:10.1101/2025.03.25.644927

bioRxiv · 10.1101/2025.03.25.644927

Yeast cluster

Braymer, J. J.·Knauer, L.·Crack, J. C.·Oltmanns, J.·Heghmanns, M.·Soares, J. C.·Le Brun, N. E.·Schünemann, V.·Kasanmascheff, M.

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Abstract

Nar1 is an essential eukaryotic protein proposed to function as an iron-sulfur (Fe/S) cluster trafficking factor in the cytosolic iron-sulfur assembly (CIA) machinery. However, such a role has remained unclear due to difficulties in purifying adequate amounts of cofactor-bound protein. The [FeFe]-hydrogenase-like protein has two conserved binding sites for [4Fe-4S] clusters, one of which is predicted to be a labile site for cluster transfer to downstream targets. Here, we report a new preparation procedure for Nar1 that facilitated studies by UV-Vis, EPR, and Mossbauer spectroscopies, along with native mass spectrometry. Nar1 recombinantly produced in E. coli contained a [4Fe-4S] cluster, bound presumably at site 1, along with an unexpected [2Fe-2S] cluster bound at an unknown site. Fe/S reconstitution reactions installed a second [4Fe-4S] cluster at site 2, leading to protein with three Fe/S cofactors. Strikingly, one [4Fe-4S] cluster was rapidly destroyed by molecular oxygen, potentially linking Nar1 oxygen sensitivity to phenotypes observed previously in vivo. These advances now allow for the pursuit of in vitro Fe/S cluster transfer assays, which will shed light on Fe/S trafficking by CIA components and how they may facilitate the insertion of [4Fe-4S] and potentially [2Fe-2S] clusters into target proteins in the cytosol.

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Braymer, J. J., Knauer, L., Crack, J. C., Oltmanns, J., Heghmanns, M., Soares, J. C., Le Brun, N. E., Schünemann, V., Kasanmascheff, M.. 2025-03-27. Yeast cluster. https://doi.org/10.1101/2025.03.25.644927

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